(a) Field of the Invention
The present invention relates to a capturing carrier for capturing air-borne microorganisms, a capturing device comprising the same, an analysis system using the same, and a method for capturing and testing microorganisms.
(b) Description of the Related Art
As a method for detecting air-borne microorganisms, a colony counting method using an agar medium, or a method for measuring turbidity using a liquid medium and the like have been known. According to these methods, air-borne microorganisms are captured in a medium and cultured for one to several days followed by a test. Accordingly, these methods have disadvantages in that it takes a long period of time for testing, and insuring biologically safety becomes difficult due to cultured microorganisms. As such, their on-line introduction to a production line of food products or pharmaceuticals is impossible. In particular, this type of method for testing microorganisms is often used for determination of microbial cleanness (i.e., germ-freeness) of an environment which requires a high level of cleanness, such as a factory for producing pharmaceuticals, a facility for processing cells, or a factory for producing food. When a culture method is used for on-site test of microorganisms under the environments, if microorganism that are amplified in a massive amount as a product of inspection process are incorporated into the site, a purpose of ensuring the cleanness of the environment cannot be preserved. As a result, there has been a problem that on-site testing cannot be easily carried out. Moreover, installation of a testing room and strict management of a products during production and carrying process are required.
Recently, in order to solve such problems, various methods have been devised by which microbial count is determined quickly and conveniently without culturing them, i.e., without a risk of biological contamination. Specifically, a method has been suggested by which constituents of a microorganism are specifically labeled by using a luminescent or a fluorescent reagent, and then luminescence and luminescence output are measured to determine the microbial biomass. As a first method, there is a method in which microorganisms contained in a sample are stained with two kinds of fluorescent reagents, and then viable and non-viable microbes are counted based on a fluorescence microscopic image analysis and the like (i.e., a fluorescent method). As a second method, ATPs are extracted from the microorganisms contained in a sample, and a biological luminescence reaction by ATP is measured by a luminometer to quantify the ATPs, and it is converted to the viable microbial count (i.e., ATP method).
However, although in principle measurement of one microbial cell is possible according to the first method, co-existence of a fluorescent contaminating material in a sample can cause an error. Further, it is not always easy to differentiate a viable cell from a non-viable cell. For instances, propidium iodide (PI), which is known for selective staining of non-viable cells, may sometimes induce lower average fluorescent intensity for non-viable cells compared to that for viable cells (“Technical Product Information “Live/Dead BacLight™ bacterial viability and counting kit (L34856)” Molecular Probes, edited version of Feb. 2, 2004”, FIG. 1C, the vertical axis represents fluorescent intensity of PI), thus there is a high chance of providing an opposite conclusion if careful attention is not paid.
According to the second method, i.e., ATP method, a molecule of ATP (adenosine triphosphate) that is a chemical contained by every living cell is taken as an indicator of the microbial count. This method takes an advantage of a biological luminescence between a luciferase as an enzyme originated from a bioorganism, and luciferin (a kind of imidazopyrazinones) as a substrate for the enzyme. Specifically, after ATPs are extracted from a sample comprising microorganisms, a mixture comprising luciferin and luciferase is added thereto for a luminescence reaction. Then, the amount of ATPs is obtained from the luminescence output, and it is converted to the amount of the microorganisms. As a result, according to this ATP method, the amount of the viable microorganisms can be determined within several minutes to several tens of minutes.
When the amount of the microorganisms is to be determined according to ATP method, an ATP eliminating agent is first added to remove free ATPs that exist outside the cell body of a viable microbe (i.e., extracellular ATPs and ATPs of non-viable microbes) and the resulting free ATPs are decomposed. Then, in order to extract ATPs from the viable cells, an ATP extracting agents is added and cell membrane of the viable microbe is destroyed to elute the ATPs contained inside the cell body of the microbe. Consequently, a luminescent reagent is added and luminescence output is measured, which is then converted to the ATP amount.
Because this ATP method is based on a biological luminescence reaction which selectively responds to ATP, in principle it is not affected by the presence of a fluorescent contaminating material. In addition, a reagent kit, which comprises an ATP eliminating agent which can remove in advance ATPs that exist outside the cell body of a viable microbe, an ATP extracting reagent for extracting ATPs from the viable microbe, and a luminescent reagent for measuring the extracted ATPs based on their biological luminescence reaction, has been commercially provided by Kikkoman Corp. (Product name; Lucifer HS set) (Journal of Japan Society for Bioscience, Biotechnology, and Agrochemistry, Vol. 78, No. 7, p. 630-635 (2004) “Application and development of a firefly luciferase” Murakami Seiji et. al). When this kit is used in conjunction with a luminometer, an influence by non-viable microbes and the like is excluded so that ATP amount solely originating from viable microbes can be selectively measured.
However, the test sample to be tested by the ATP method is a microorganism that is dispersed in an aqueous solution and its application to air-borne microbes has not been suggested. Meanwhile, for carrying out a test based on a method for culturing air-borne microbes, a product which comprises a collecting apparatus in an impactor style and includes an installable medium cassette carrying a capturing carrier that consists of an agar (polysaccharide) culture medium in a gel phase is commercially available (i.e., M Air T type air sample manufactured by Millipore Company). However, an air sampler which can be applied for the ATP method is not commercially available. When the ATP method is applied for testing air-borne microbes, it can be considered that an air sampler in an impactor style for a culture method is used for capturing microbes in a capturing carrier, the captured microbes are recovered from the capturing carrier and dispersed in a suspension, and the microbial counting is carried out based on the ATP method using a reagent kit for detecting a biological luminescence reaction, such as Lucifer HS set.
However, the capturing carrier which consists of a gel phase agar medium and is used for an air sampler in a conventional method (i.e., a combination method for an air sampler and the ATP method) is used under the purpose of culturing captured microbes on the capturing carrier itself. As such, a process of recovering the microbes from the capturing carrier (herein after, referred to as a “recovery process”) has not been considered in the conventional method. For the recovery process, there are problems as follows. Specifically, a gel phase capturing carrier is advantageous for capturing the microbes, however, taking out the microbes from the capturing carrier is difficult, so that recovery rate of microbes is poor. Meanwhile, if an agar gel is heated, it can be transformed into a sol phase which can be handled practically the same as a microbial suspension.
However, since high temperature of more than 80° C. is required for the transformation of an agar gel to a sol, most of viable microbes would be damaged or killed during the process. As a result, there are problems that recovery rate of microbes is poor and viable microbes and non-viable microbes cannot be differentiated from each other. Depending on the types of bacteria, inactivation or cell death may occur at the temperatures above 40° C. Under the circumstances, in order to increase the recovery rate of viable microbes, a mild condition which includes repetition of adding a solution having the temperature near the room temperature is added to a gel without transforming an agar gel into a sol and recovering the microbes that are transferred to the solution as microbial suspension is necessary. However, this method still requires a time-consuming and delicate human work by a skilled person, and it cannot be automated, has poor reproducibility and a risk of giving a false positive result due to contamination originating from human workers.
Meanwhile, although a great amount of a solution can be used for recovery to simplify working processes, when the amount of microbial suspension is large, microbial count per unit volume of liquid (i.e., concentration) becomes lower so that the use efficiency of the microbes during measuring process will be consequently lower. For example, the amount of a sample liquid for carrying out the protocols (previous protocols) of Lucifer HS set by Kikkoman Corp., which is the representative product for the ATP method, is 0.1 mL.
Therefore, when the volume of microbial suspension is 1 mL, use efficiency of the suspension during the measurement process is 10%, and it will be 1% when the volume is 10 mL, indicating that the use efficiency of the suspension becomes lower as the volume of the microbial suspension increases. As such, this method has a problem that convenience and use efficiency cannot be achieved at the same time.
In addition, only 1/10 of a sample liquid is actually used for luminescence measurement according to the conventional protocol, there is a problem that overall use efficiency is again only 10% of the recovery rate of the microbes. Further, there is another problem that when extremely small amounts of air-borne microbes (i.e., several to several tens of microbes) are to be measured in a highly clean environment such as a clean room, low recovery rate or low use efficiency may cause an omission error in microbial counting, yielding a false negative result. In order to avoid such omission error, sampling volume of an air sample should be increased and other measures should be also taken. As a result, problem still remains that sampling time is long and results cannot be obtained in short time.
As such, if the above described method is automated as it is, a system construction becomes complicated and a solution required for recovery should be used in more excessive amount to leave a margin, resulting in even lower use efficiency. In addition, when this method is applied to an actual sample, a great amount of contaminants are included in a collected sample in addition to microbes. In this regard, the conventional method is problematic in that a reaction for eliminating ATP, a reaction for extracting ATP, and an ATP luminescence reaction are affected by the contaminants so that measurement accuracy is poor.
In addition, some of microorganisms are present in a state of a spore which is wrapped by a spore membrane having high endurance to chemical substances or heat. Thus, various reactions including the ATP method cannot easily occur. In order to solve such problems of a spore-forming microbe, it has been suggested that a germination inducing factor is first added and then germination is allowed to occur, followed by carrying out various further reactions. For example, according to Japanese Patent Application National Publication (Laid-Open) No. 2001-511356, it is described that germination is allowed to occur in a culture medium comprising a germination factor. In addition, according to Japanese Patent Application Laid-Open (JP-A) No. 2002-330740 and JP-A No. 2004-2229, it is described that germination inducing factor is added first and then germination of a microbe is allowed to occur, followed by sterilization. According to Japanese Patent Application National Publication (Laid-Open) No. 2005-516213, it is described that a germination inducing factor is added and species-specific cell spores are detected and quantitated. According to JP-A No. 2006-174751, it is described that by adding alanine to a cell, strength of the spores of a spore forming microbe is reduced to facilitate the counting of spore forming microbes. According to JP-A No. 2005-253365, a method is described by which microorganisms are admixed with a germination inducing factor and then genes are eluted from them.
Thus, to have a reaction with spore, addition of a germination inducing factor is effective and even for the test based on the ATP method, it is expected that adding germination inducing factor is an effective thing to do.
However, for the ATP method in which various reagents including an ATP eliminating agent, an ATP extracting agent, and a luminescent reagent are added for the reaction, further addition of a germination inducing factor can make the test more cumbersome and can yield a longer test time. Especially, as the reaction with a germination inducing factor takes a relatively a long period of time, the test time becomes longer, making its on-line introduction to a production line difficult.
In addition, when a subject to be tested by the ATP method is an air-borne microorganism, it should be captured in a capturing carrier and then later recovered from the capturing carrier. Time is also required for such process, thus it is problematic in that a long period of time is required from capturing to testing.